This application relates to a method and kit for high-resolution, DNA-based typing of the three classical HLA Class I genes, namely the HLA-A, HLA-B, and HLA-C loci, in a patient sample.
The HLA Class I genes comprise the three classical genes encoding the major transplantation antigens HLA-A, HLA-B, and HLA-C and seven other Class I genes of which HLA-E, HLA-F and HLA-G are probably functional genes and HLA-H, HLA-I, HLA-K and HLA-L are pseudogenes. All classical Class I genes have a similar structure: Exon 1 (73 bp)--Intron 1 (130 bp)--Exon 2 (270 bp)--Intron (272 bp)--Exon 3 (276 bp)--Intron 3 (588 bp). The classical Class I genes are highly polymorphic. As of 1996, at least 82 alleles of HLA-A, 186 of HLA-B and 42 of HLA-C had been identified.
Methods for determining HLA-A, HLA-B, and HLA-C alleles in a patient sample have been heavily investigated because of the functional importance of these genes in transplant tissue matching and autoimmune diseases. Serological tests including the complement-dependent cytotoxicity assay (see Terasaki and McClelland, Nature, 204:998, (1964)) providing low resolution identification of HLA type have been most commonly applied to date. Unfortunately, such low resolution tests cannot detect and distinguish all functionally significant transplant antigens (Anasetti et al. Hum. Immunol., 29:70 (1990)).
High resolution tests are more desirable because they lead to improved tissue matching and reduced transplant rejection. Current methods of high resolution typing include using Sequence-Specific Oligonucleotide Probes (SSOP). The SSOP technique is well illustrated in U.S. Pat. No. 5,451,512 assigned to Hoffman-La Roche, Inc. Using a reverse dot blot format, HLA-A probes are immobilized on a membrane, and the labeled target (patient sample) DNA is hybridized to the membrane-bound probe as described in Saiki et al., 1989, Proc. Natl. Acad. Sci. 86:6230-6234. The sites of hybridization are detected, and HLA-A type can be deduced. This method does not involve direct DNA sequencing.
Another high resolution test is the Amplification Refractory Mutation System (ARMS) (see "HLA Class I SSP ARMS-PCR typing kit" Reference manual, June 1995 edition. Imperial Cancer Research Fund). Since primers which do not match target nucleic acid are not amplified under stringent hybridization conditions, the use of primers with specifically designed 3'-ends for amplification allows the allelic composition to be determined on the basis of which ARMS primers fail to amplify, and which ones are successful. This method does not involve direct DNA sequencing.
A direct DNA sequencing method for HLA Class I typing has been proposed by Santamaria et al ("HLA Class I Sequence-Based Typing" Hum. Immunol. 37, 39-50 (1993); WO 9219771; U.S. Pat. No. 5,424,184). This method has identified oligonucleotide primers for amplification and sequencing of the classical Class I genes. The method suffers because it focuses on cDNA (exon) sequences which, because of the sequence diversity, offer a very limited selection of conserved primer hybridization sites. Further, the sites disclosed were determined before the recent discovery of dozens of more alleles that now need to be considered in identifying HLA type. In addition, because the Santamaria sequencing primers hybridize within an exon, they do not provide information for DNA sequence upstream of the primer which is potentially decisive for distinguishing among alleles.
Intron sequences, as disclosed in the instant patent application, provide the preferred hybridization sites for amplification and sequencing primers for the HLA-A, HLA-B and HLA-C genes. Intron sequences for an HLA Class I gene were disclosed at least as early as 1985. (Weiss et al Immunobiol 170:367-380, (1985)). Due to the substantial diversity, and the difficulties in sequencing, few intron sequences have been published subsequently. A report of Blasczyk and Wehling (in Abstracts of The American Society for Histocompatability and Immunogenetics 22nd Annual Meeting, Oct. 11-15, 1996) states that intron sequences have been determined in 48 well-defined cell lines and 195 PCR-typed clinical samples.
Cereb et al. (Tissue Antigens 1995: 45:1-11), undertook the identification of intron sequences useful for locus-specific amplification primer sets for all Class I genes. Amplified fragments were characterized by SSOP and no direct sequencing of the amplified fragments was performed.
Blasczyk et al. have disclosed primers for use in amplification of the HLA-A locus in preparation for sequence-based typing of a sample. Tissue Antigens 47: 102-110 (1996). Twenty-one different primer mixes having different serologically defined specificities were identified for amplification of the second and third exons of the HLA-A gene. The resulting amplification products were then analyzed by sequencing.
Johnston-Dow et al (Poster Presentation: 1995 ASHI Meeting, Dallas, Tex.) presented a system for direct sequence determination of HLA-A wherein degenerate exon-based primers were used to amplify exons 1 to 5 of the genomic HLA-A DNA sequence. Sequencing of the amplified fragment was obtained using degenerate primers which hybridize to intron regions flanking exons 2 and 3.
Direct sequencing of HLA-B alleles from an amplified genomic DNA fragment was performed by Petersdorf and Hansen. (Tissue Antigens 1995 46: 73-85). A total of five primer sets (ten different primers) with different allelic specificities were used to amplify genomic material, and the amplification products were then sequenced.
Notwithstanding the various efforts which have been made to develop effective methods for typing HLA genes, none of the methods previously disclosed actually provides a test which will permit rapid typing of HLA alleles using a minimum number of reagents in a manner which is reproducible and which, in ordinary use, actually achieves typing of all allelic variants. Thus, for example, certain primers may theoretically be capable of achieving amplification and subsequent sequencing of all allelic variants of one of the classical HLA genes, yet in practice reactions with these primers are found to suffer from dropouts which preclude the specific detection of some alleles. There remains, therefore, a need for a method and kit which obtain direct high-resolution sequence information for the HLA-A, HLA-B and/or HLA-C genes using a minimum of oligonucleotide primers for all alleles of the gene analyzed.
It is an object of the present invention to provide a method and kit for direct sequencing of the HLA-A, HLA-B, and/or HLA-C alleles comprising a minimum number of oligonucleotides which hybridize to intron regions of these genes and which between them can amplify all alleles of these genes.
It is a further object of the present invention to provide a method and kit for direct sequencing of the HLA-A, HLA-B, HLA-C alleles using a minimum number of oligonucleotides which hybridize to intron regions of these genes and which between them can sequence all alleles of these genes.